1. Field of the Invention
This invention relates to assay methods which involve the separation of glycosylated from nonglycosylated hemoglobins by ion exchange column chromatography, and specifically to means for correcting the results of such assays for variations in temperature. The measurement of glycosylated hemoglobin serves as a time-averaged indicator of blood glucose levels in patients afflicted with diabetes mellitus.
The level of hemoglobin A.sub.1 (HbA.sub.1), a glycosylated form of adult hemoglobin (HbA), is known to be higher in the blood of diabetic persons than in that of normal persons. Hemoglobin A.sub.1, is composed of at least three subfractions, HbA.sub.1a, HbA.sub.1b, and HbA.sub.1c. These three subfractions account for about seven percent of the total hemoglobin in the normal adult with HbA.sub.1c being the major subfraction. Each of these fractions contributes to the observed increase. Therefore, assays for HbA.sub.1 in the aggregate as well as those specific for HbA.sub.1c are used to monitor carbohydrate control in diabetic patients. Ion exchange separation techniques which isolate the hemoglobin component or components of interest from the remaining components have been found to be particularly useful in this regard. A weak cation exchange column is exemplary of columns used in tests of this kind. A buffer solution which preferentially elutes the HbA.sub.1 fraction is passed through the resin, leaving nonglycosylated hemoglobins behind.
Unfortunately, the efficiency of the separation is temperature dependent. Resins for analytical use are thus formulated and prepared with a distinct operating temperature in mind, herein designated a "design" or "reference" temperature, which typically ranges from about 22.degree. C. to about 24.degree. C. At temperatures below the design temperature, an increasing amount of the fraction of interest (be it HbA.sub.1 or HbA.sub.1c) remains in the resin, whereas at higher temperatures increasing amounts of nonglycosylated hemoglobins elute off the resin in combination with the fraction of interest. To achieve comparison among samples on a common basis, therefore, it has been necessary to either impose strict temperature control to the design temperature or to use temperature conversion charts which require a highly accurate measurement of the temperature of columns and reagents during the performance of the assay.
In view of the inconvenience of such measures, it is desirable to find a method for improving the accuracy of ion exchange column chromatographic techniques for the measurement of HbA.sub.1 values which avoids both the need for highly precise temperature measurements and control.
2. Description of the Prior Art
A general discussion of glycosylated hemoglobins and their relevance to diabetes mellitus is offered by Bunn, et al., Science, 200, pp. 21-27 (1978). The use of ion exchange resins is described by Chou, et al., Clin. Chem., 24 (10), pp. 1708-1710 (1978) and in a series of U.S. Pat. Nos. to Acuff: 4,142,855, 4,142,856, 4,142,857 and 4,142,858 (all issued on Mar. 6, 1978), 4,168,147 (issued on Sept. 18, 1979) and 4,238,196 (issued on Dec. 9, 1980).
Lyophilized hemoglobin preparations stabilized with polyhydroxyl compounds are disclosed by Proksch, et al., Am. J. Clin. Pathol., 74(1), pp. 64-67 (1980), Bonderman, et al., Clin. Chem., 26(2), pp. 305-308 (1980).